Strikingly novel instrumentation for electron paramagnetic resonance (EPR) spectroscopy at the high microwave frequency of 94 GHz (W-band) has been developed. The broad long-term objective of the present proposal is to establish usefulness of this instrument in a significant biomedical application: the study of cholesterol-mediated lipid interactions using dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol across the phase diagram. Phospholipids, as well as cholesterol, spin-labeled at a number of available sites using either 14N or 15N isotopes will be used. Measurements of bimolecular collision rates that occur between a 14N-tagged site and a 15N-tagged site provide information about the impact of cholesterol on molecular dynamics. The time scale for the experimental methods is on the order of 10 s, which is a typical value for the spin-lattice relaxation time, T1, of the spin label. Measurements in the range of 10 times faster to 10 times slower are within reach. This is a range that is considered not only to be of high biological relevance but also to be essentially inaccessible using other instrumental modalities. Two complementary EPR techniques will be used: saturation recovery (SR) and pulse electron-electron double resonance (ELDOR). Excitation in both techniques will be an adiabatic rapid sweep of an intense microwave frequency across a selected region of the EPR spectrum. This is a CHIRP frequency-swept pulse. It is a highly innovative technical approach that requires the use of an arbitrary waveform generator (AWG) and a broadband loop-gap resonator (LGR). Specific aims are as follows: (1) Development of CHIRP excitation and observation methods for SR followed by measurements across the experimental parameters of the phase diagram using available spin labels. (2) Use of the data of Specific Aim 1 to design analogous ELDOR experiments, which will also be applied across the phase diagram using various spin-label pairs-one with 14N and one with 15N. Measurements of collision frequencies between labels at different depths-so-called vertical fluctuations-will be made to provide insight into the mediation of membrane dynamics by cholesterol. The diffusion-in-a-cone model will be tested using 14N/15N pairs, each member of the pair at the same depth, as a function of increasing depth, across the phase diagram. And (3), which is an intense state-of-the-art engineering initiative: direct digital detection of the microwave carrier in the existing instrument. A novel aspect of Aim 3 is the use of magnitude detection of SR and ELDOR signals-which is new in the context of EPR and is particularly appropriate when using CHIRP pulses. This proposal rests on the highly significant hypothesis that molecular structures require knowledge of molecular dynamics to be biologically relevant. Extension of the methodology to membrane-bound proteins is foreseen. PUBLIC HEALTH RELEVANCE: Molecular dynamics occur in all biological molecules across a wide range of motional frequencies, some of which are central to biological function. The nitroxide-radical spin-label method, based on electron paramagnetic resonance (EPR), is suitable for the study of molecular dynamics, including dynamics with characteristic motional periods of a millisecond to a microsecond that are biologically- relevant. The proposal applies a new EPR spectrometer that has many innovative features and operates at the high microwave frequency of 94 GHz to characterize cholesterol-mediated lipid interactions in membranes as a function of cholesterol content and temperature.